Polish Journal of Microbiology2012, Vol. 61, No 1, 51–55

ORIGINAL PAPER

Introduction

Cellulase is an enzyme that hydrolyzes the β-1,4-glycosidic bonds in cellulose to release glucose units (Nishida et al., 2007). It has been generally accepted that e"ective biological hydrolysis of cellulose into glu-cose requires the synergistic actions of three enzymes including endo-β-1,4-glucanase (EG; EC 3.2.1.4, ran-domly cleaving internal linkages), cellobiohydrolase (CBH; EC 3.2.1.91, specifically hydrolyzing cellobiosyl units from non-reducing ends), and β-D-glucosidase (EC 3.2.1.21, hydrolyzing glucosyl units from cello-oligosaccharides) (Perez et al., 2002). As for carboxym-ethyl cellulose (CMC), only endoglucanases are needed to degrade it (Robson and Chambliss, 1989). Cellulases have many industrial applications from the generation of bioethanol, a realistic long-term energy source, to the finishing of textiles (Ando et al., 2002; Lynd et al., 2002), formulation of washing powders, extraction of fruit and vegetable juices, and starch processing (Camassola and Dillon, 2007). Many Bacillus sp. including B. brevis (Singh and Kumar, 1998), B. pumilus (Kotchoni et al., 2006), B. amyoliquefaciens DL-3 (Lee et al., 2008), and Bacillus subtilis YJ1 (Li-Jing Yin et al., 2010) have been exploited for cellulase production. In this study an attempt has been made to screen carboxymethyl cellu-

lase (CMCase)-secreting Bacillus sp. from a paddy field located in the southwestern region of India. #e main objective of the present study was to isolate potential CMCase-secreting organisms for purification of cellu-lase by convenient methods. A study of this kind will improve our knowledge on the biotechnological applica-tion of bacteria found in the natural environment.

Experimental

Materials and Methods

Isolation of bacteria from a paddy field for CMCel-lulase secretion. A small amount of soil from paddy field was collected at Nagercoil, Kanyakumari (India). Suitable dilutions were made and the bacteria were cul-tured in the nutrient agar medium composed of in (g/l): peptic digest of animal tissue, 5.0; beef extract, 1.5; yeast extract, 1.5; sodium chloride, 5 and agar, 15. #e pH was adjusted to 7.0 with the addition of 1N NaOH. #e organisms were incubated at 37°C for 24 h.

Screening and identification of CMCase-secreting organism. For the screening of CMCelluase produc-tion, the bacterial isolates were streaked on CMC agar medium (g/l) (peptic digest of animal tissue, 5.0; beef extract, 1.5; yeast extract, 1.5; sodium chloride, 5.0,

Purification and Characterization of Carboxymethyl Cellulasefrom Bacillus sp. Isolated from a Paddy Field

PONNUSWAMY VIJAYARAGHAVAN1* and S.G. PRAKASH VINCENT2

1 Centre for Marine Science and Technology, Manonmaniam Sundaranar University,Rajakkamangalam 629 502, Kanyakumari District, Tamilnadu, India

2 International Centre for Nanobiotechnology, Centre for Marine Science and TechnologyManonmaniam Sundaranar University, Rajakkamangalam 629 502, Kanyakumari District, Tamilnadu, India

Received 20 February 2011, revised 14 December 2011, accepted 20 December 2011

A b s t r a c t

A microorganism hydrolyzing carboxymethyl cellulose was isolated from a paddy field and identified as Bacillus sp. Production of cellulase

by this bacterium was found to be optimal at pH 6.5, 37°C and 150 rpm of shaking. #is cellulase was purified to homogeneity by the com-

bination of ammonium sulphate precipitation, DEAE cellulose, and sephadex G-75 gel filtration chromatography. #e cellulase was puri-

fied up to 14.5 fold and had a specific activity of 246 U/mg protein. #e enzyme was a monomeric cellulase with a relative molecular mass

of 58 kDa, as determined by SDS-PAGE. #e enzyme exhibited its optimal activity at 50°C and pH 6.0. #e enzyme was stable in the pH

range of 5.0 to 7.0 and its stability was maintained for 30 min at 50°C and its activity got inhibited by Hg2+, Cu2+, Zn2+, Mg2+, Na2+, and Ca2+.

K e y w o r d s: Bacillus sp., biological degradation, carboxymethyl cellulase, paddy field, cellulase purification

* Corresponding author: P. Vijayaraghavan, Centre for Marine Science and Technology, Manonmaniam Sundaranar University,

Rajakkamangalam-629 502, Kanyakumari District, Tamilnadu, India; e-mail: venzymes@gmail.com

Vijayaraghavan P. and Vincent P. 152

agar, 15, CMCellulose, 10) and incubated at 37°C for 48 h. A%er incubation, bacterial growth was seen as a  single line on the plate. To visualize the hydrolysis zone, the agar medium was 'ooded with an aqueous solution of Gram’s iodine. Gram’s iodine forms a blu-ish-black complex with cellulose but not with hydro-lyzed cellulose, giving a sharp and distinct zone within 3–5 min (Kasana et al., 2008). Bacterial identification was conducted based on ‘Bergey’s Manual of Systematic Bacteriology’ (Jones and Collins, 1984).

Microorganism and submerged fermentation. #e Bacillus sp. was cultured in the nutrient medium com-posed of in (g/l): peptic digest of animal tissue, 5.0; beef extract, 1.5; yeast extract, 1.5; sodium chloride, 5.0 and CMCellulose, 10 (pH 7.0), and sterilized at 121°C for 20 min. A loopful culture of 18 h Bacillus sp. was inoc-ulated into the nutrient medium with rotary shaking at 150 rpm at 37°C for 36 h. #e culture medium was centrifuged at 6000×g for 20 min and passed through a 0.22-μm membrane (Rankam, NY0213SF) to remove the cells. #e resulting supernatant was used for further CMCase purification.Enzyme activity assays:a) Endo-β-1,4-glucanase. Endo-β-1,4-glucanase (EG)

activity was determined by incubation of 1.0 ml of 0.2% (w/v) CMC in 0.025 M phosphate bu"er (pH 7.0) with 1.0 ml of appropriate concentration of enzyme at 37°C. A%er 30 min of reaction, 1 ml of dinitrosalicylic acid (DNS) was added and boiled in a water bath for 5 min to stop the reaction. #e resulting samples were then cooled to room tempera-ture and the absorbance measured at 540 nm (A

540).

One unit of EG activity was defined as the amount of enzyme that could hydrolyze CMC and release 1 μg of glucose within 1 min at 37°C (Nelson, 1944).

b) Avicelase. Avicelase activity was determined by incubation of 1.0 ml of 0.2% avicel (w/v) in 0.025 M phosphate bu"er (pH 7.0) with 1.0 ml of appropri-ate concentration of enzyme at 37°C. A%er 30 min of reaction, the activity was measured. One unit of avicelase activity was defined as the amount of enzyme that could hydrolyze avicel and release 1 μg of glucose within 1 min at 37°C (Nelson, 1944).

c) Filter paperase. In this assay Whatman No. 1 filter paper was used as the substrate. Fi%y milligrams of filter paper was dissolved in 0.025 M phosphate bu"er (pH 7.0) and 1.0 ml of appropriate concentra-tion of enzyme. #e mixture was incubated at 37°C for 30 min and the reaction was terminated by add-ing 2 ml of DNS. One unit of filter paperase activ-ity was defined as the amount of enzyme that could hydrolyze filter paper and release 1 μg of glucose within 1 min at 37°C (Nelson, 1944).Growth and fermentation. A loopful culture of

the bacterial isolate was inoculated into the minimal

medium (g/l) (peptic digest of animal tissue, 5.0; beef extract, 1.5; yeast extract, 1.5; and sodium chloride, 5.0 (pH 6.5)), and incubated at 37°C in an orbital shaker at 150 rpm.  Five millilitres of the culture medium was withdrawn at regular 12 h intervals and the cell den-sity determined at 600 nm (Henriette et al., 1993) up to 72 h. To determine the optimum fermentation period for enzyme production the culture medium was centri-fuged at 6000×g for 10 min. #e culture supernatant was assayed for determining enzyme activity (Nelson, 1944).

Determination of protein concentration. #e pro-tein concentration of the crude enzyme as well as that of the purified enzyme was determined by the method of Lowry et al. (1951) using bovine serum albumin (BSA) as the standard.

Substrate specificity. #e hydrolytic ability of the crude enzyme sample against 0.2% CMC, 0.2% avicel, 0.2% cellobiose, and filter paper (20 mg) in 0.025 M phosphate bu"er (pH 7.0) was determined to evaluate its substrate specificity.CMCase purification

Ammonium sulphate precipitation. #e proteins in the crude preparation were precipitated by the addi-tion of solid ammonium sulphate at 40% to 80% satu-ration. #e precipitate was allowed to form at 4°C for 24 h, and was collected by centrifugation at 5000 ×g in a cold centrifuge for 20 min. #e precipitate was redis-solved in 10 ml of bu"er A (0.025 M sodium phosphate bu"er, pH 7.0 containing 0.001 M ethylene diaminetet-raacetic acid (EDTA) and 0.001 M 2-mercaptoethanol).

Purification by ion exchange chromatography on diethylaminoethyl (DEAE) cellulose. DEAE cel-lulose was packed into a vertically mounted column (1.5×15 cm) and the 'ow rate of the column was set at 0.5 ml/min. #e column was equilibrated with 4 bed volumes of bu"er A. #e enzyme concentrate obtained from ammonium sulphate precipitation was redissolved in a minimal amount of bu"er A and dialysed over-night against the same bu"er. #e column was washed to remove all unbound proteins and a  linear gradient of 0 to 0.5 M NaCl-added bu"er A was used to elute the bound proteins. Fractions (2 ml) were collected at a 'ow rate of 0.5 ml/min. Fractions exhibiting cellulase activity were pooled and concentrated with ammonium sulphate. #e precipitate was collected by centrifugation at 5000 × g in a refrigerated centrifuge at 4°C for 20 min, redissolved in bu"er A and dialysed against the same bu"er for 4 h.

Gel filtration on Sephadex G-75. Sephadex G-75 slurry was packed into a column (0.6 × 50 cm) and equilibrated with bu"er A. #e fractions with the high-est cellulase activity from the ion exchange chromatog-raphy step were applied to the column. Fractions (2 ml) were collected at a 'ow rate of 0.25 ml/min. #e eluate was monitored for protein concentration at 280 nm and

Carboxymethyl cellulase from Bacillus sp.1 53

fied organism was Gram-positive, motile, rod-shaped, starch-hydrolyzing, and nitrate-, catalase- and urease-positive. #e organism was seen to produce acid when fructose, galactose, mannitol, sucrose, trehalose, and cellobiose were added to it. Based on morphological and biochemical characteristics, the organism was ten-tatively identified as Bacillus sp. according to Bergey’s Manual of Systematic Bacteriology.

Growth curve and fermentation. #e Bacillus sp. attained maximum growth a%er 36 h of fermentation and the absorbance was 1.69 at 600 nm. #e absorb-ance declined to 1.38, 1.14, and 0.836 at 48, 60, and 72 h of incubation, respectively. #e enzyme secretion increased to its maximum of 13 U/mg protein at 36 h of fermentation. Enzyme production was found to be 3.8, 7.1, 7, 3.1 and 0.7 U/mg protein at 12, 24, 48, 60 and 72 h of fermentation, respectively (Fig. 1).

E"ect of pH on enzyme production. E"ect of pH on enzyme production was studied by culturing the organism at various pH levels (5.5–8.0) using 1 N HCl/NaOH. Enzyme production was 3.6, 5.1, 9.6, 11.1, and 1.4 U/mg protein at pH 5.5, 6.0, 7.0, 7.5, and 8.0, respec-tively. Enzyme activity was found to be high at pH 6.5 (14.4 U/mg protein) (Fig. 2).

Purification of cellulase from the Bacillus sp. #e enzyme was purified through 3 steps including ammo-nium sulphate fractionation, DEAE cellulose and gel filtration column chromatography. #e recovery and purification were 24% and 14.5-fold, respectively, a%er

Fig. 1. E"ect of fermentation period on CMCellulase

by Bacillus sp. in submerged fermentation

Fig. 2. E"ect of pH on CMCellulase from Bacillus sp.

was also assayed for enzyme activity. Fractions with high enzyme activity were pooled and precipitated with solid ammonium sulphate. Again, the precipitate was collected by centrifugation at 5000 × g at 4°C for 30 min, redissolved in a minimal amount of bu"er A and then dialysed against the same bu"er overnight. #e purified enzyme thus obtained was stored at –20°C.

SDS-PAGE and molecular mass determination. Denaturing sodium dodecyl sulphate/polyacrylamide gel electrophoresis SDS/PAGE (10%) was performed to determine the molecular mass of the cellulase following the methods of Laemmli (1970). #e molecular weight of the subunit of the enzyme was estimated with phos-phorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (43 kDa), and carbonic anhydrase (29 kDa) markers. #e sample obtained from the gel filtration column was loaded into the gel. Following electropho-resis, the gel was documented using a gel documenta-tion system (Syngene, UK) and the molecular weight of the purified cellulase determined.

E"ect of pH on enzyme activity and stability. #e optimum pH for the activity of the purified enzyme was determined by using the following bu"ers (0.1 M): citrate bu"er (pH 4.0); succinate bu"er (pH 5.0–6.0); sodium phosphate bu"er (pH 7.0); tris bu"er (pH 8.0); glycine-NaOH bu"er (pH 9.0). #e stability of the enzyme at various pH was examined by incubating the enzyme solution with the above-mentioned bu"-ers at 37°C for 30 min prior to incubation with sub-strate. Enzyme activity was assayed as described by Nelson (1944).

E"ect of temperature on enzyme activity and stability. #e temperature profile of the purified cel-lulase enzyme was determined by performing the rou-tine enzyme assay at di"erent temperatures: 30, 40, 50, 60, 70 and 80°C. To determine thermal stability, the crude enzyme was incubated (without substrate at increasing temperatures, from 30 to 80°C) for 30 min and cooled. Enzyme activity was assayed as described by Nelson (1944).

E"ect of divalent ions on enzyme activity. To study the e"ect of divalent ions on enzyme activity, the enzyme sample was incubated with various diva-lent ions namely, Ca2+, Mg2+ , Zn2+ , Cu2+ , Na2+ , Hg2+ at 0.005 M concentration for 30 min. Enzyme activity was assayed as described by Nelson (1944).

Results

Screening and identification of cellulolytic bac-teria from a paddy field. Five cellulase-secreting bac-terial strains were isolated from a paddy field using spread plate technique on basal medium. Among the five bacterial strains, Bacillus sp. showed the highest cellulolytic activity on the CMC agar plate. #e identi-

Vijayaraghavan P. and Vincent P. 154

Sephadex G-75 chromatography. #e purification pro-cedure of cellulase is summarized in Table I.

Molecular mass of Bacillus sp. cellulase. SDS-PAGE analysis of the purified enzyme revealed a single band with a molecular mass of 58 kDa (Fig. 3). #e enzyme was electrophoretically run at pH 8.3 on a 11% acryla-mide gel and stained with Coomassie Brilliant Blue R-250. #is was associated with cellulase activity.

E"ect of pH on enzyme activity and stability. In Bacil-lus sp. cellulase activity was found to be high (19 U/mg protein) at pH 6.0. Enzyme activity was 4.1, 7.5, 15, 5.5, and 2.0 U/mg protein at pH 4.0, 5.0, 7.0, 8.0, and 9.0, respectively (Fig. 4). #is enzyme was highly stable at pH 6.0 at which the relative enzyme activity was 100%. #e relative enzyme activity was 27%, 73%, 67%, 42%, and 13% at the pH 4.0, 5.0, 7.0, 8.0, and 9.0, respectively.

E"ect of temperature on cellulase activity and stability. #e e"ect of cellulase activity was studied by incubating the reaction mixture at various temperatures

(30–80°C). Enzyme activity was found to be high (24 U/mg protein) at 50°C. Enzyme activity was 12.5, 18, 16, 7, and 1.5 U/mg protein at 30, 40, 60, 70, and 80°C, respectively (Fig. 5). #e enzyme activity was also high (100%) at the denaturizing temperature of 50°C and this indicated that it retained its activity a%er sample denaturation for 30 min. #e relative enzyme activity was 67%, 83%, 80%, 35%, and 4% at 30, 40, 60, 70, and 80°C respectively.

E"ect of divalent ions on cellulase activity. #e e"ect of divalent ions on enzyme activity was investi-gated by allowing the divalent ions (0.001 M) to react with the cellulase sample. Among divalents, Mn2+ enhanced the relative enzyme activity (124%) when compared with the control (100%). Ca2+, Cu2+, Hg2+, Mg2+ Na2+, and Zn2+ inhibited the enzyme activity and the relative activity was 88%, 47%, 13%, 73%, 56%, and 81%, respectively.

Discussion

Screening of cellulase-secreting organism. In the present study five cellulase-secreting bacterial strains were isolated from a paddy field. Based on cellulolytic activity, the organism which showed more zone (3 cm) was used for CMCase production and the organism was tentatively identified as Bacillus sp. Growth curve was plotted for this species. CMCase activity was meas-ured during growth in the basal medium, in which the

Crude extract 95 3 285 17 1

Amm. sulphate 15 9 136 31 1.8

DEAE-cellulose 12 0.68 8.2 144 8.5

Sephadex G-75 8 0.43 3.5 246 14.5

Table I

Summary of purification of CMCase from Bacillus sp.

Purification

step

Vol.

(ml)

Total amt

of protein

(mg)

S. acti-

vity

(U/mg)

Purifi-

cation

(fold)

Protein

content

(mg/ml)

Fig.4. E"ect of pH on purified CMCase from Bacillus sp.

Fig. 3. SDS/PAGE of purified CMCase from Bacillus sp. M:

Protein markers; A: Purified cellulase

Fig. 5. E"ect of temperature on purified CMCase from Bacillus sp.

Carboxymethyl cellulase from Bacillus sp.1 55

enzymatic activity increased up to 36 h of fermentation, before declining gradually (Fig. 1). Similar results were obtained with Bacillus subtilis YJ1 (Yin et al., 2010).

E"ect of pH on enzyme production. Cellulase production was found to be high at pH 6.5 when the enzyme activity was 14.4 U/mg protein (Fig. 2). Simi-lar results were obtained with Bacillus licheniformis-1 (Dhillon et al., 1985) and with Bacillus coagulans (Odeniyi et al., 2009).

Substrate specificity analysis. #e crude enzyme was found to have the highest activity on CMC (16.5 U/mg protein). Significant activity was also observed with the crystalline forms of cellulose such as avicel, filter paper and cellobiose. Similar results were obtained with two other bacterial strains, Bacillus CH43 and HR68 (Mawadza, 2000).

Purification of CMCase. #e crude protein was first precipitated with 40% to 80% ammonium sulphate saturation. Following concentration, DEAE cellulose ion exchange chromatography led to the partial purification of the enzyme. #e fractions having cellulase activity were pooled and concentrated and loaded on a sepha-dex G-75 gel filtration column. #e final sephadex G-75 gel filtration chromatography step yielded 246 U/mg protein of electrophoretically pure CMCase from the culture supernatant (Fig. 3). Similar chromatographic methods were employed for the purification of cellu-lase from Bacillus subtilis YJ1 (Yin et al., 2010). SDS/PAGE revealed that the molecular mass of the purified CMCase was 58 kDa. Similar result was obtained with Bacillus amyoliquefaciens DL-3 (Lee et al., 2008).

E"ect of pH on enzyme activity and stability. #e purified cellulase had optimum activity at pH 6.0. Similar results were obtained with Bacillus subtilis YJ1 (Yin et al., 2010). #is enzyme was highly stable at pH 6.0, which was similar to Bacillus CH43 and HR68 (Mawadza et al., 2000).

E"ect of temperature on cellulase activity and sta-bility. #e cellulase isolated from the Bacillus sp. was highly active at 50°C (24 U/mg protein) and highly sta-ble at this temperature for at least 30 min of enzyme denaturation. Similar results were found with Bacillus CH43 and HR68 (Mawadza et al., 2000).

E"ect of divalent ions on cellulase activity. Cellulase was highly active in the presence of 0.005 M Mn2+, and Hg2+ strongly inhibited enzyme activity. Similar results were reported for Bacillus subtilis YJ1 (Yin et al., 2010) and Bacillus amyoliquefaciens DL-3 (Lee et al., 2008).

#e progress in biotechnology of CMCellulase is remarkable and attracting worldwide attention. Over - production of CMCase from Bacillus sp. can be achieved by physical, chemical methods of mutagenesis and through recombinant DNA technology. #is kind of study also helps to produce CMCase in commercial scale utilizing local agro-wastes in solid state culture.

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